Antibiotics, and in general, antimicrobials, have been in commercial use for decades. Antimicrobials have played an enormous role in both enhancing quality of life and extending life expectancy.
Despite their effectiveness in fighting infection, antimicrobial agents have some well-known drawbacks. First, they can eliminate significant numbers of mutually beneficial native flora, opening the door for opportunistic infection. In other words, the successful treatment of unwanted bacteria or other microorganism may kill enough mutually beneficial microorganisms to allow an opportunistic pathogen to increase numbers to a pathogenic level.
For example, humans have over 400 species of commensal bacteria, present mostly in the colon and ileum, whose existence is essential to normal digestion by humans. Intestinal bacteria alone collectively weigh as much as one kilogram and number approximately 1014. Yet, humans manage to cohabit with the intestinal flora in a mutually beneficial symbiotic relationship. Centers for Disease Control: “Campaign to Prevent Antimicrobial Resistance in Healthcare Settings,” 2002. Mutually beneficial gut flora compete with pathogenic species for space and nutrients, usually preventing pathogenic colonization. Similarly, mutually beneficial bacteria, if allowed to grow unchecked, may themselves become pathogenic.
On the other hand, an antibiotic that kills large numbers of mutually beneficial bacteria may eliminate competition for pathogenic bacteria, providing them with space and nutrients that otherwise would be unavailable to them. One example is the opportunistic pathogen is Clostridium difficile. Typically, C. difficile is not present at pathogenic levels in the intestine. If, certain mutually beneficial intestinal bacteria are eliminated after intense or prolonged antibiotic exposure, C. difficile will begin to colonize the intestine in large numbers, resulting in a significant infection capable of producing toxins that result in inflammation and injury to the intestinal lining. This colonization is allowed as a result of the lack of other bacteria, bacteria whose existence would have checked the growth of C. difficile, that have been eliminated by the long term antibiotic treatment.
Another common example is seen in the treatment of any number of infections in women. Infectious conditions, upper respiratory infections or urinary tract infections for example, treated with antibiotics may lead to other complications. Often times, women treated with broad spectrum antibiotics for a variety of bacterial infections often develop vaginal yeast infections due to the wiping out of the natural flora of the vagina, a flora that normally keep yeast, Candida albicans specifically, in check. A natural antibiotic treatment that does not disrupt the balance of the normal flora is needed.
The normal flora is easily distinguished from pathogenic bacteria, as are normal flora that have mounted an opportunistic infection. Antibiotics are created to selectively kill a certain group of bacteria, Gram positive for example. Upon the discovery and development of a new antimicrobial, an antimicrobial's spectrum of action is easily determined by using well-established techniques. The Kirby-Bauer Disc Diffusion test is one such technique. Microbes are grown in agar plates containing paper discs coated with the antimicrobial agent. By measuring the diameter of growth inhibition around the disc, one can determine which strains are susceptible or resistant to the antimicrobial agent. Discs with larger diameters of inhibition indicate that the strain is susceptible to the antimicrobial agent, and likely will be easily treated in a patient setting. Discs with smaller diameters of inhibition surrounding them are indicative of the presence of more resistant microbes that may take longer to kill in a patient. Those with no diameter of inhibition indicate that the microbes either already harbor resistant genes or have mutated to become resistant. As resistant bacteria propagate, the genes responsible for their resistance will be passed to successive generations. If resistant bacteria establish an infection in a patient, the results can be devastating.
Antimicrobial resistance has been recognized since the introduction of penicillin nearly 50 years ago when patients having penicillin-resistant S. aureus infections appeared. As early as 1946, only 3 years after companies began mass-producing penicillin, a London hospital reported that 14% of S. aureus strains taken from patients were penicillin-resistant. Drexler, M., “Secret Agents: The Menace of Emerging Infections,” Joseph Henry Press, 2002. Today, hospitals worldwide are facing infection control problems from the rapid emergence and dissemination of other microbes resistant to one or more antimicrobial agents.
Microbes, regardless of their resistance status, are normally recognized and ablated by an individual's immune system. If, however, the microbe population is sufficiently large and their growth outpaces the immune system's ability to eliminate them, an infection can result that may threaten the health of the individual. Treatment with an antimicrobial agent will eliminate a large percentage of the pathogenic microbial population in a patient, which allows the patient's immune system to ablate the remaining pathogenic organisms. In contrast, resistant microbes do not succumb to antibiotic therapy and if the immune system is unable to eliminate them, their colonization may result in persistent infections that are difficult, if not impossible, to treat using currently available therapies.
Prolonged antibiotic exposure, among other causes, may also result in “super infection.” Super infection is best understood with reference to basic Darwinian theory. Those microbes whose phenotype presents more resistance to certain antimicrobials will result in the proliferation of similar bacteria who are “selected in” by the antimicrobial. Essentially, the more sensitive bacteria are killed while the more resistant survive and thrive. Antimicrobials are designed to kill off sensitive bacteria, bacteria that have proliferated for any number of reasons, beyond the body's cellular and humoral based immunity systems' ability to overcome the infection. Usually, the number of sensitive bacteria significantly outnumber the resistant strains. Antimicrobial therapy directed to a specific microbe, in addition to the body's immune system, has been successful in clearing infection. However, long term administrations of an antibiotic treatment, osteomylitis for example, may result in the killing of all sensitive bacteria but no resistant bacteria. In this case, if the proliferation of resistant bacteria overcomes the body's natural ability to control the rate of growth of the resistant bacteria, a super infection of resistant bacteria develops.
In a nearly opposite situation, that of uncompleted antimicrobial treatment, a similar resultant super infection is possible. Antibiotic therapy is designed to last beyond the period of symptomatic treatment. In other words, the medications are to be taken beyond that point a patient no longer experiences symptoms of the infection. When treatment is aborted prior to a full “course of antibiotics,” a super infection may result. The early portion of a normal course of antibiotic treatment usually results in the killing of the majority of sensitive bacteria. The remaining course of antibiotics keeps the remaining bacteria in check while the body fights the remaining sensitive bacteria and the rare population of resistant bacteria, a population that may not exist in every patient. In those cases where a patient may contain a population of resistant bacteria, the uncompleted course of antibiotic treatment may result in a proliferation of resistant bacteria beyond the body's natural ability to fight infection. Essentially, the incomplete course kills the weakest bacteria leaving the strong to survive. In Darwinian terms, the resistant bacteria have been selected in. As more and more hosts select in more and more resistant strains of bacteria, these bacteria become predominant and new medications are needed to combat them. A treatment that poses less of a risk of creating resistant strains of bacteria is needed.
Resistant microbes represent a major concern to the medical community. More than an estimated $30 billion was spent in 2000 alone treating antimicrobial-resistant infections. “Antimicrobial Resistance,” Office of Communications and Public Liaison, National Institute of Allergy and Infectious Diseases, National, Bethesda, Md., June 2000. Standard therapies for treating infections become more limited in the face of antimicrobial-resistant bacteria, increasing the risk of serious, untreatable, sometimes life-threatening infections. Drug-resistant pathogens are a growing threat and are troublesome in healthcare settings. Nearly 2 million patients contract hospital-acquired infections, or “nosocomial” infections, every year in the United States alone, and about 90,000 die as a result of their infection. Centers for Disease Control: “Campaign to Prevent Antimicrobial Resistance in Healthcare Settings,” 2002. More than 70% of the bacteria that cause hospital-acquired infections are resistant to at least one of the drugs commonly used to treat them. Persons infected with drug-resistant organisms are more likely to have longer hospital stays and/or require treatment with second or third-choice drugs that may be less effective, more toxic, and/or more expensive.
A recent and publicized example of this phenomenon occurred with the drug ciprofloxicin and the bacteria, Psudomonas aeriginosa. P. aeriginosa is a Gram negative bacteria that is resistant to many antimicrobials. It has been especially difficult to treat in patients with diabetic infections, which commonly lead to gangrene, amputation and death. The drug was used as specific therapy for patients having a P. aeriginosa infection and for broad spectrum treatment in cases where the pathogen had not been identified. The drug was successful at treating infection and doctors across the country began using the medication as a broad-spectrum therapy, especially in the common cases of upper respiratory infection. In time, ciprofloxicin-resistant strains of P. aeriginosa emerged. An infection with these resistant bacteria posed a risk to the patient and an increased difficulty of treatment for the physician. This problem is now difficult in an immunocompromised patient.
A similar example involves another genus of bacteria common to humans. Staphylococci are a type of Gram positive bacteria normally present in skin and mucosal membranes of the body. S. aureus, in particular, is a virulent opportunistic pathogen that causes many skin, bone, mucous membrane infections, bacterial endocarditis, respiratory infection, food poisoning and toxic shock syndrome, to name only a few. S. aureus infections were commonly treated with the methicillin, a member of the penicillin class of antibiotics. This was the treatment of choice before beta lactamase inhibitor antibiotics, clavulanic acid for example, became available. Although methicillin was effective against “Staph” infections, some S. aureus strains developed resistance to it, and only a few antibiotics were available to successfully treat methicillin-resistant Staphylococcus aureus (MRSA). One such antibiotic commonly used to treat MRSA infection is vancomycin. A strain of S. aureus, however, with reduced susceptibility to vancomycin (VISA) has already been identified. Khurshid, M. A., et. al., Staphylococcus aureus with Reduced Susceptibility to Vancomycin—Illinois, 1999, Morbidity and Mortality Weekly Report, 48(51): 1165–1167 (2000). Strains of S. aureus resistant to methicillin and other antibiotics are endemic in hospitals. Infection with methicillin-resistant MRSA strains may also be increasing in non-hospital settings. Increasing reliance on vancomycin has led to the emergence of vancomycin-resistant enterococci (VRE), bacteria that infect wounds, the urinary tract and other sites. Until 1989, such resistance had not been reported in U.S. hospitals. By 1993, however, more than 10 percent of hospital-acquired enterococci infections reported to the CDC were resistant. Nordenberg, T., Miracle Drugs Versus Superbugs, FDA Consumer Article, November-December 1998.
Streptococcus pneumoniae is another pathogenic bacteria. It causes thousands of cases of meningitis and pneumonia, and 7 million cases of ear infection in the United States each year. Currently, about 30 percent of S. pneumoniae isolates are resistant to penicillin, the primary drug used to treat this infection. Many penicillin-resistant strains are also resistant to other antimicrobial drugs. “Antimicrobial Resistance,” Office of Communications and Public Liaison, National Institute of Allergy and Infectious Diseases, National, Bethesda, Md., June 2000.
In sexually transmitted disease clinics that monitor outbreaks of drug-resistant infections, doctors have found that more than a third of gonorrhea isolates are resistant to penicillin, tetracycline, or both. “Sexually Transmitted Disease Surveillance 1997” Division of STD Prevention, September 1998, U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Center for HIV, STD, and TB Prevention, Division of STD Prevention, Atlanta, Ga.
Strains of multi-drug-resistant tuberculosis (MDR-TB) have also emerged over the last decade and pose a particular threat to people infected with HIV. MDR-TB strains are just as contagious as TB strains that are drug-sensitive. MDR-TB is more difficult and vastly more expensive to treat, and patients may remain infectious longer due to inadequate treatment, thereby increasing the likelihood of transmission. “Antimicrobial Resistance,” Office of Communications and Public Liaison, National Institute of Allergy and Infectious Diseases, National, Bethesda, Md., June 2000.
Diarrheal diseases cause almost 3 million deaths a year, mostly in developing countries, where resistant strains of highly pathogenic bacteria such as Shigella dysenteriae, Campylobacter, Vibrio cholerae, Escherichia coli and Salmonella are emerging. Recent outbreaks of Salmonella food poisoning have occurred in the United States. A potentially dangerous “superbug” known as Salmonella typhimurium, resistant to ampicillin, sulfa, streptomycin, tetracycline and chloramphenicol, has caused illness in Europe, Canada and the United States. “Antimicrobial Resistance,” Office of Communications and Public Liaison, National Institute of Allergy and Infectious Diseases, National, Bethesda, Md., June 2000.
Fungal pathogens account for a growing proportion of nosocomial, or hospital acquired, infections. Fungal diseases such as candidiasis and Pneumocystis carinii pneumonia are common among AIDS patients, and isolated outbreaks of other fungal diseases in people with normal immune systems have occurred recently in the United States. Scientists and clinicians are concerned that the increasing use of antifungal drugs will lead to drug-resistant fungi. In fact, recent studies have documented resistance of Candida species to fluconazole, a drug used widely to treat patients with systemic fungal diseases. “Antimicrobial Resistance,” Office of Communications and Public Liaison, National Institute of Allergy and Infectious Diseases, National, Bethesda, Md., June 2000. Moreover, recently isolated C. albicans strains from patients have exhibited resistance to amphotericin B, which is currently the only antifungal agent from the class of approximately 200 known polyene agents safe enough for intravenous administration. Ellis, D., “Amphotericin B: Spectrum and Resistance,” Journal of Antimicrobial Chemotherapy 49 Supp 1: 7–10, 2002.
In recent years, the emergence of resistant strains to commonly used antimicrobials has stimulated a search for new, and naturally occurring, antimicrobial compounds having clinical utility. Thus, a new and natural medication is needed that uniformly attacks bacteria that are both sensitive and resistant to classical medicines. Further, the new medication should be equipotent across genera of bacteria to help, as closely as possible, the immune system to maintain the intricate balance of the body's natural flora.
Certain endogenous peptides have shown antimicrobial activity against bacteria, fungi and enveloped viruses but with little or no cytolytic activity, have been isolated from diverse sources. Martin, E et al, “Defensins and other endogenous peptide antibiotics of vertebrates,” J. of Leukocyte Biology 58: 128–36, 1995. Most of these peptides share the property of being cationic but they differ considerably in some features, such as their size, the presence of disulfide bonds and structural motifs. Gabay, J., “Ubiquitous Natural Antibiotics,” Science 264:373–4, 1994. These peptides have been shown to exert their antimicrobial activities either by forming multimeric pores in the lipid bilayer of the cell membrane or through interacting with macromolecular synthesis after penetration into the cell membrane. Zasloff, M., “Antibiotic peptides as mediators of innate immunity,” Current Opinions in Immunology 4: 3, 1992; see also Boman, H. et al, “Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine,” Infection and Immunity 61: 2978–84, 1993. The most important aspect of antimicrobial peptides is that they rarely induce bacterial resistance. Oren, Z., et al, “A class of highly potent antibacterial peptides derived from pardaxin, a pore-forming peptide isolated from Moses sole fish Pardachirus marmoratus,” European Journal of Biochemistry 237, 303–10, 1996. Accordingly, antimicrobial peptides are promising candidates in the continuing search for a new class of antibiotics. It is an object of this invention to create antimicrobial peptides for use in antimicrobial treatments.